In recent years, in response to the problem of global warming caused by petroleum-derived carbon dioxide, opportunities continue to arise throughout the world to overhaul social structures that are overdependent on fossil fuels. This trend is leading to increasingly active operation of “biorefineries” that make use of bioprocessing technology, for which research is accelerating throughout the world, but unfortunately under the current state of affairs no research results have yet been obtained for biosynthesis of aromatic compounds, although in light of the importance of aromatic compounds including aniline derivatives for the chemical industry, diligent efforts are being expended in research toward synthesis of aromatic polymers.
For example, PTL 1 discloses a technique relating to polymer synthesis using 4-aminocinnamic acid (4ACA) which is a natural molecule, and reports that a high heat-proof polymer is obtained from 4-aminocinnamic acid.
Also, as disclosed in NPL 1, the metabolic pathway for biosynthesis of 4-aminophenylalanine (4APhe) via shikimic acid has been elucidated (see p. 2818, FIG. 1), but there has been no disclosure nor teaching of ammonia-lyase functioning in an organism and converting 4-aminophenylalanine to 4-aminocinnamic acid.
NPL 2 describes isolation of the gene for phenylalanine ammonia-lyase of the yeast Rhodotorula glutinis JN-1 (hereunder abbreviated as “Rgpal”), depositing of the yeast at CCTCC (China Center For Type Culture Collection) as deposit number M2011490, and creation of an optimum pH mutant by site-specific mutagenesis of the gene. Furthermore, since the Chinese Patent Application specification of which the authors of NPL 2 are the inventors (hereunder, PTL 2) was published on Apr. 24, 2013, the actual sequence of Rgpal is publicly known. However, it is not disclosed that the enzyme can produce 4-aminocinnamic acid using 4-aminophenylalanine as the substrate.
Thus, 4-aminophenylalanine (4APhe) is an important substance in that it is a precursor for 4-aminocinnamic acid (4ACA).
Also, NPL 3 discloses, as shown in FIG. 1, conversion of chorismic acid to 4-amino-4-deoxychorismic acid (ADC) by PapA (4-amino-4-deoxychorismic acid synthase), conversion of ADC to 4-amino-4-deoxyprephenate (ADP) by PapB (4-amino-4-deoxychorismic acid mutase), and conversion of ADP to 4-aminophenylpyruvic acid by PapC (4-amino-4-deoxyprephenate dehydrogenase).
Also, it is believed that 4-aminophenylpyruvic acid is converted to 4-aminophenylalanine (4APhe) by the action of microbial endogenous enzymes.
In addition, PTL 3 discloses that biosynthesis of 4-amino-4-deoxychorismic acid (ADC), at least catalyzed by an enzyme belonging to the class of aminodeoxychorismic acid synthases, is carried out by in vivo fermentation in a host microorganism having 4-amino-4-deoxychorismic acid synthase at an increased level of activity, while obtaining a fermentation culture broth comprising 4-amino-4-deoxychorismic acid (ADC) and 4-amino-4-deoxyprephenate (ADP), and that the compounds are recovered from the fermentation culture broth, either together or each one separately.
However, when the conventionally known pap genes, i.e. the 3 key enzymes known in pathways of antibiotic production (for example, PapA, PapB, PapC of Streptomyces venezuelae) are simply utilized directly, the productivity of 4-aminophenylalanine (4APhe) by fermentation is no more than about 0.2 g/L, and even attempting various combinations of conventionally known pap genes, it accumulates at no more than about 0.9 g/L.
Such low productivity has been an obstacle when trying achieve industrial mass production of aniline derivatives including 4-aminophenylalanine (4APhe), 4-aminocinnamic acid (4ACA), 2-(4-aminophenyl)aldehyde, 4-aminophenylacetic acid and 4-aminophenethylethanol (4APE), from carbon sources such as glucose by fermentation (see FIG. 1).
Thus, a method allowing industrial mass production of aniline derivatives including 4-aminophenylalanine (4APhe), 4-aminocinnamic acid (4ACA), 2-(4-aminophenyl)aldehyde, 4-aminophenylacetic acid and 4-aminophenethylethanol (4APE) from carbon sources such as glucose by fermentation has not yet been established, and there is strong demand to develop one.